Abstract
Purpose: :
PF-655, a synthetic 19-mer siRNA targeting the RTP801 gene, is currently in Phase 2 trials for the treatment of wet AMD and DME. Preclinical studies have shown a dose-related suppression of RTP801 expression in rat CNV and DME models. Investigative studies were conducted with PF-655 to validate Dutch Belted rabbit as a biologically relevant species for gene silencing to support continual dosing studies.
Methods: :
Cross-species gene comparison and DNA sequencing was used to determine the level of homology between PF-655 and rabbit RTP801 gene. RNA from primary rabbit retina was used to access RTP801 gene expression in vivo and obtain a limit of detection for the assay. Human (293T) and rabbit (SIRC cornea) cell lines were stimulated with CoCl2 to mimic hypoxic stress (an inducer of RTP801 expression) and treated with PF-655. Q-PCR and immunoblot analysis was performed to gauge RTP801 expression.
Results: :
DNA sequence analysis showed a one base mismatch between PF-655 and the sequence from primary retina tissue and a rabbit cell line. RTP801 mRNA was detected in rabbit retina and cycle threshold numbers were within a valid range for robust detection of the gene in vivo and subsequent gene silencing down to 10% of control. SIRC and 293T CoCl2-stressed cells induced RTP801 expression 30-40-fold above control conditions. Cells treated with 50-100 nM PF-655 showed a decrease in RTP801 expression (~40-50%) relative to appropriate control treated cells.
Conclusions: :
These results support our investigation into cross-species validation of gene suppression by a therapeutic siRNA designed to a human gene. With a one-base mismatch, PF-655 silenced the RTP801 gene in a rabbit ocular cell line to a comparable level with that of a human cell line. Sequence analysis and expression data confirmed the relevance of the RTP801 target gene in rabbit and the utility of the rabbit as a relevant species model for RTP801 modulation in ocular efficacy and/or safety studies. Additionally our work outlines a tractable method to validate relevant larger non-rodent species for ophthalmic drug testing.
Keywords: gene/expression • hypoxia • drug toxicity/drug effects